Small molecule inhibition of a PDZ-domain interaction

ABSTRACT

Novel compounds that have been found effective in inhibiting PDZ domain interactions, and particularly interactions of PDZ domains in MAGIs with the oncogenic (tumor suppressor) protein PTEN and interactions between the PDZ domain in the Dishevelled (Dvl) protein and other proteins such as the Frizzled (Fz) protein, have the general formula  
                 
The invention also includes combinatorial libraries, arrays and methods for screening and studying proteins using such compounds. Compounds of the invention have produced apoptosis in certain cell lines that overexpress the Dishevelled protein (Dvl); inhibiting Wnt signaling.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the priority of U.S. provisional application60/463,198 filed Apr. 15, 2003, the entire contents of which are herebyincorporated herein.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

This work was supported in part by National Institutes of Health grantno. GM31497. The government of the United States of America may havecertain rights in this invention.

BACKGROUND AND FIELD OF THE INVENTION

This invention relates to the inhibition of interactions of PDZ domainsof a protein or proteins with other proteins, and more particularly tonovel compounds that have been found to be effective in inhibiting PDZdomains. In a very specific aspect, this invention relates to theinhibition of a PDZ domain of proteins that regulate the function of theoncogenic protein PTEN, or of the PDZ domain of the Dishevelled protein(Dvl), and compounds that have been found to possess such inhibitingcapability. Compounds of the invention have been shown to produceapoptosis in cancer cells overexpressing Dvl.

PDZ domains are regions of signaling proteins that function to modulateprotein-protein interactions such as protein-protein recognition. PDZdomains were named for three proteins in which this domain was initiallydiscovered: PSD-95 (a 95 kDa protein involved in the signaling at thepost-synaptic density), DLG [Drosophila lethal (1) discs large-1], andZO-1 (the zonula occludens-1 protein involved in maintenance ofepithelial polarity). These proteins play important roles in neuronalsynaptic transmission, tumor suppression, and cell junction formation,respectively. They are understood as functioning in vivo by organizingmultiprotein complexes that function in signaling, e.g. communicationbetween cells. For example, PDZ-organized signaling complexes are knownto function in communication involving neurons or epithelial cells,e.g., by coupling activated receptors to downstream second messengersystems, and in transporting and targeting proteins to sites of cellularsignaling. They are involved in the functioning of important cell signalmediators including ion channels, transmembrane receptors, andregulatory enzymes. PDZ-containing proteins are believed to be involvedin disorders associated with defective cell signaling, includingischemic nerve damage and tumorigenesis.

Structurally, PDZ domains are 80-90 amino acid modular proteininteraction domains that comprise six beta-strands (betaA to betaF) andtwo alpha-helices, A and B, compactly arranged in a globular structure.Peptide binding of the ligand takes place in an elongated surface grooveas an anti-parallel beta-strand interacts with the betaB strand and theB helix. The structure of PDZ domains allows binding to a freecarboxylate group at the end of a peptide through a carboxylate-bindingloop between the betaA and betaB strands.

Among proteins with PDZ domains are the MAGIs (membrane associatedguanylate kinase proteins with inverse orientation). Proteins in thisclass participate in the assembly of multiprotein complexes on the innersurface of the plasma membrane at regions of cell-cell contact. TheMAGIs are a small family of adaptors, widely expressed in the humanbody, that have six PDZ domains. MAGI-3 binds to the tumor suppressorPTEN, a lipid/protein phosphatase, using its second PDZ domain(MAGI3-PDZ2). The interaction of MAGI-3 and PTEN decreasesphosphotidylinositol 3-kinase and Akt/PKB signaling whereas release ofPTEN from MAGI-3 increases Akt/PKB signaling. Normally, Akt/PKBsignaling ensures cell survival during response to cellular insults bysuppressing apoptosis. However, PTEN mutants that cause constitutiveAkt/PKB signaling have been associated with human cancers. Chemicaldisruption of this interaction would be a unique way to investigate therole Akt/PKB signaling in transformation and cancer, and could affectthe development of cancerous growths.

Also among proteins that have been found to possess a PDZ domain is theDishevelled protein (Dvl). Its interactions with the Wnt and Frizzledproteins have been indicated as being involved in one or more types ofcancers.

Small-molecule inhibitors of interactions of proteins having PDZ domainswith other proteins would be desirable.

BRIEF SUMMARY OF THE INVENTION

This invention relates to novel compounds that have been found effectivein inhibiting PDZ domain interactions, and particularly interactions ofPDZ domains in MAGIs with the oncogenic (tumor suppressor) protein PTEN,and of a PDZ domain in the Dishevelled protein with Frizzled proteins,and to the inhibition of PDZ domain activity by the use of thesecompounds.

The novel compounds have the general formula

in which:

-   -   R₀ is selected from the group consisting of C₁-C₃ alkyl,        cyclopropyl, halo, OR₅ and S(O)_(m)R₅ in which n is 0, 1 or 2;    -   R₁ and R₂ are independently selected from the group consisting        of C₂-C₈ alkenyl, phenylcyclopropyl, phenylpropenyl,        R₆—X₂—C(R₈)(R₈)—R₇—; and R₆—X₂—N(R₈)—R₇—;    -   R₃ and R₄ are independently hydrogen, methyl or ethyl;    -   R₅ is methyl or ethyl;    -   R₆ is selected from the group consisting of hydrogen, C₁-C₁₀        alkyl, aryl, W, Y, NH₂, NHCONR₃R₄, NHCOOR₃ and NHSO₂R₉;    -   R₇ is selected from the group consisting of a direct bond, an        alkyl group having from 1 to 10 carbon atoms, aryl,        —(NH)_(p)(CH₂CH₂O)_(q)(NH)_(p)— in which p is 0 or 1 and q is an        integer from 1 to 4, and W;    -   R₈ is selected from the group consisting of H, Y, OH,        —NHCONR₃R₄; —NHCOOR₃; —NHSO₂R₉, —(CH₂)_(r)CO₂R₃, and        (CH₂)_(r)CO₂NR₃R₄ in which r is an integer from 1 to 3;    -   R₉ is aryl or C₁-C₆ alkyl;    -   X₁ is —CH—, —C-hal, —C(CH₃) or —C(C₂H₅), in which hal stands for        a halogen atom (preferably chloro, fluoro or bromo);    -   X₂ is selected from the group consisting of a direct bond, —NH—,        —N(CH₃)—, —NCONR₃R₄—, —NCOOR₃—, and NSO₂R₉;    -   W is a saturated carbocyclic or heterocyclic group;    -   Y is selected from the group consisting of COOH, COOR₃, CONR₃R₄,        CONHSO₂R₅, hydroxymethyl, —CH₂COOH, CH₂CONR₃R₄; and        5-tetrazolyl; and    -   Z is —CH₂—, —CH(CH₃)—, C(CH₃)₂— or —CO—;    -   and hydrates and salts thereof, and labeled derivatives thereof.

This invention also includes processes and intermediates for thepreparation of these compounds. This invention also includes librariesof such compounds and methods for preparing such libraries. Theinvention also includes labeled versions of the compounds and chemicalprobes prepared by linking the novel compounds to various labelingmoieties.

In another aspect, this invention includes methods for inhibiting theinteraction of a PDZ domain of a protein with other proteins, moreparticularly the interaction of a MAGI protein or of the Dishevelledprotein (Dvl) with other proteins, by contacting the protein thatcontains the PDZ domain, or the PDZ domain, with an inhibitory effectiveamount of a compound as defined herein. In yet another aspect thisinvention relates to methods of studying protein interactions and/or PDZdomain functioning that involves contacting the protein or the domainwith a compound or compounds as described herein. The invention alsoincludes arrays of such compounds for studying protein interactions orfor screening proteins for PDZ domain activity.

In a more specific aspect of this, the invention comprises inhibitinginteractions of PDZ domains in MAGIs with the oncogenic (tumorsuppressor) protein PTEN or of inhibiting interactions between the PDZdomain in the Dishevelled protein and other proteins, for example theFrizzled (Fz) protein, by contacting the MAGI protein, the Dishevelledprotein, or a PDZ domain of such protein with an effective inhibitoryamount of a compound as described herein.

In another embodiment this invention also provides therapeutic methodsof treating cancer comprising contacting the cancer or cancerous cellswith an effective PDZ domain-inhibiting amount of a compound of theinvention. In these embodiments, the cancer cell typically is in apatient and the step of contacting is carried out by administering atherapeutic agent, namely one or more compounds of the invention, or acomposition containing the same, to the patient. The method may furthercomprise administering to the patient a second therapeutic agent, suchas a chemotherapeutic agent or radiation therapy. The cancer cell may bea breast cancer cell, colorectal cancer cell, a lung cancer cell, asarcoma cell, or a mesothelioma cell, a prostate cancer cell, apancreatic cancer cell, a cervical cancer cell, an ovary cancer cell, agastric cancer cell, an esophageal cancer cell, a head and neck cancercell, a hepatocellular carcinoma cell, a melanoma cell, a glioma cell, asquamous cancer cell, or a glioblastoma cell.

U.S. patent application Ser. No. 10/678,639 filed Oct. 3, 2003, titled“Methods for Treating Cancer by Inhibiting WNT Signaling”, of He et al.,is hereby incorporated herein in its entirety. That applicationdiscloses inhibiting the growth of cancer cells that overexpress a Wntprotein by contacting the cell with an agent that inhibits binding ofthe Wnt protein to a Frizzled receptor. PCT application WO 02/088081discloses that overexpression of Wnt appears connected to the occurrenceof head and neck squamous cancer. Wong et al, J. Mol. Cell 12.1251(November, 2003) disclosed that binding of the Dlv protein to Fz occursat the PDZ domain of Frz. Inhibition of the PDZ-domain/Dlv interactioncan thus inhibit Wnt signaling and consequently inhibit the growth ofcancer cells

The tern “Frizzled protein” (Fz or Fzd) refers to a family of mammalianproteins related to the Drosophila frizzled genes, which play a role inthe development of tissue polarity. The Frizzled family comprises atleast 10 mammalian genes. Exemplary human Frizzled receptors includeFrizzled1, Frizzled2, Frizzled3, Frizzled4, Frizzled5, Frizzled6,Frizzled7, Frizzled8, Frizzled9 and Frizzled10. Frizzled proteinreceptors are involved in a dynamic model of transmembrane signaltransduction analogous to G-protein-coupled receptors withamino-terminal ligand binding domains.

The term “Dishevelled” or “Dvl” refers to a member of a family ofDishevelled proteins, the full-length sequences of which typicallypossess three conserved domains, a DIX domain, present in the Wntantagonizing protein Axin; a PDZ domain involved in protein-proteininteractions, and a DEP domain found in proteins that regulate RhoGTPases. Dvl proteins include, for example, Dvl-1, Dvl-2, and Dvl-3.Nucleic acid and protein Dvl sequence are known from a variety ofspecies, including mouse and human. Exemplary human Dvl-1, Dvl-2, andDvl-3 protein sequences are available under reference sequencesNP_(—)004412, NP_(—)004413, and NM_(—)004414, respectively.

“Inhibitors of Wnt signaling” refers to compounds that, e.g., bind toDishevelled proteins so as to interfere with Dishevelled/Frizzledinteraction, and consequently partially or totally block Wnt signaling,as measured for example, in known assays for Wnt signaling (e.g.,measurement of y catenin levels, or oncogene expression controlled byTcf and Lef transcription factors).

A “cancer cell that overexpresses a Wnt protein” is a cancer cell inwhich expression of a particular Wnt protein is at least about 2 times,usually at least about 5 times the level of expression in a normal cellfrom the same tissue. Methods for determining the level of expression ofa particular gene are well known in the art. Such methods includeRT-PCR, use of antibodies against the gene products, and the like.

In still another aspect this invention involves screening proteins forPDZ domain activity by contacting the proteins with a compound orcompounds of the invention.

Studying the functioning of proteins having a PDZ domain or screeningproteins for PDZ domain activity may involve the use of a singlecompound of the invention, or a number, including a large number ofcompounds of the invention. The latter may be carried out with a numberof tests using individual proteins, or with arrays or libraries of suchcompounds. The compounds may be in the above-shown form or preferablyalso include a labeling moiety. Additionally, the compounds may beconjugated or bonded to a solid support, for example a plate or aparticulate material, and such combinations of solid support andcompounds are another aspect of this invention.

By “inhibitors” is meant compounds that, e.g., bind to, partially ortotally block stimulation, decrease, prevent, or delay activation, orinactivate, desensitize, or down-regulate signal transduction.Similarly, the term “inhibition” means a partial or total blocking,stimulation, decrease, prevention or delaying of activation, orinactivation, desensitizing or down-regulation of signal transduction.An “effective inhibitory amount” of a compound or composition is anamount that produces inhibition in a particular assay or in a patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the overlay of the PTEN sequence TKV with compounds ofthis invention.

FIG. 2 depicts fluorescence polarization competition of a compound ofthe invention for the PDZ2 binding site on protein GST-MAGI-3.

FIG. 3 depicts the effect of two compounds of the invention on PKBactivity.

FIG. 4 depicts the results of an evaluation of the irreversiblecharacter of a compound of the invention.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to novel compounds that have been found effectivein inhibiting PDZ domain interactions, particularly interactions of aPDZ domain in MAGIs with the oncogenic (tumor suppressor) protein PTENand interactions of a PDZ domain in the Dishevelled protein with aFrizzled protein or receptor, and to the inhibition of PDZ domainactivity by the use of these compounds.

The novel compounds have the general formulas:

in which

-   -   R₀ is selected from the group consisting of C₁-C₃ alkyl,        cyclopropyl, halo, OR₅ and S(O)_(m)R₅ in which m is 0, 1 or 2;    -   R₁ and R₂ are independently selected from the group consisting        of C₂-C₈ alkenyl, phenylcyclopropyl, phenylpropenyl,),        R₆—X₂—C(R₈)(R₈)—R₇—; and R₆—X₂—N(R₈)—R₇—;    -   R₃ and R₄ are independently hydrogen, methyl or ethyl;    -   R₅ is methyl or ethyl;    -   R₆ is selected from the group consisting of hydrogen, C₁-C₁₀        alkyl, aryl, W, Y, NH₂, NHCONR₃R₄, NHCOOR₃ and NHSO₂R₉;    -   R₇ is selected from the group consisting of a direct bond, an        alkyl group having from 1 to 10 carbon atoms, aryl,        —(NH)_(p)(CH₂CH₂O)_(q)(NH)_(p)— in which p is 0 or 1 and q is an        integer from 1 to 4, and W;    -   R₈ is selected from the group consisting of H, Y, OH,        —NHCONR₃R₄; —NHCOOR₃; —NHSO₂R₉, —(CH₂)_(r)CO₂R₃, and        (CH₂)_(r)CO₂NR₃R₄ in which r is an integer from 1 to 3;    -   R₉ is aryl or C₁-C₆ alkyl;    -   X₁ is —CH—, —C-hal, —C(CH₃) or —C(C₂H₅), in which hal stands for        a halogen atom (preferably chloro, fluoro or bromo);    -   X₂ is selected from the group consisting of a direct bond, —NH—,        —N(CH₃)—, —NCONR₃R₄—, —NCOOR₃—, and NSO₂R₉;    -   W is a saturated carbocyclic or heterocyclic group;    -   Y is selected from the group consisting of COOH, COOR₃, CONR₃R₄,        CONHSO₂R₅, hydroxymethyl, —CH₂COOH, CH₂CONR₃R₄; and        5-tetrazolyl; and    -   Z is —CH₂—, —CH(CH₃)—, C(CH₃)₂— or —CO—;    -   and hydrates and salts thereof, and labeled derivatives thereof.

Some preferred embodiments for R₁ and R₂ include C₃-C₈ alkyl; C₃-C₆cycloalkyl; C₃-C₈ alkenyl; —(CH₂)_(m)C₆H₅ where m is 0 or an integerfrom 1-3; —CH₂OC₆H₅, CH₂COC₆H₅, phenyl(C₂-C₄ alkenyl), or analogousmoieties having substituted phenyl groups; optionally substitutedphenylcyclopropyl; —(CH₂)_(s)OH, —(CH₂)_(s)CONH₂ and —(CH₂)_(s)COOHwhere s is an integer from 1 to 3; phenyl; thienyl; and optionallysubstituted C₃-C₆ cycloalkyl-(C₁-C₃ alkyl). Particularly preferredembodiments are phenethyl, hydroxyethyl, n-butyl and n-pentyl.

In one preferred embodiment, the compounds are of formula (I), R₁ isother than hydroxyethyl or a phenethyl group, and R₂ is not an alkylgroup optionally substituted by hydroxy. In another preferred embodimentthe compounds are of formula (II), R₁ is other than an alkyl group, R₂is not a phenethyl group and Z is CO.

The term “alkyl” as used herein means a straight or branched chain, ornon-aromatic cyclical, hydrocarbon radical, or combination thereof, thatis fully saturated and has the number of carbon atoms designated (i.e.C₁-C₁₀ means one to ten carbon atoms). Examples of acyclic alkyl groupsinclude, but are not limited to, groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, homologs and isomersof, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.Examples of cyclical alkyl groups include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, and the like. The term “lower alkyl” means agroup of the type mentioned, having up to ten, preferably up to six,carbon atoms. For use in the invention, alkyl groups generally may be ofany desirable size. Preferably they will contain up to 20, morepreferably, up to 10, and most preferably up to 8, carbon atoms.

The term “alkenyl” as used herein means a straight or branched chain, ornon-aromatic cyclical, hydrocarbon radical, or combination thereof, thatcontains one or more olefinic bonds and has the number of carbon atomsdesignated (i.e. C₁-C₁₀ means one to ten carbon atoms). Examples ofacyclic alkenyl groups include, but are not limited to, groups such asvinyl, allyl, propenyl, butenyl, crotyl, butadienyl, and the like.Examples of cyclical alkyl groups include cyclopentenyl, cyclohexenyl,cyclohexadienyl, and the like. For use in the invention, alkenyl groupsgenerally may be of any desirable size. Preferably they will contain upto 20, more preferably, up to 10, and most preferably up to 8, carbonatoms.

The alkyl and alkenyl groups used in this invention may be unsubstitutedor may be mono- or polysubstituted, as indicated above.

Substituted alkyl, alkenyl, or cycloalkyl groups also include arylalkylgroups, namely alkyl (including cycloalkyl) groups substituted by one ormore aryl groups; for instance, benzyl, phenethyl, triphenylmethyl,phenylpropenyl, cyclohexylmethyl, cyclopropylmethyl, and the like. Theyalso may include cycloalkyl groups having an aryl group as a substituentsuch as phenylcyclopropyl. The aromatic ring or rings in the arylalkylgroups may be further substituted similarly to other aliphatic groups,e.g. chlorobenzyl, methylbenzyl, etc. Substituted alkyl groups alsoinclude alkyl groups substituted by one or more saturated or unsaturatedheterocyclic groups, i.e. the substituted alkyl groups arepyridylmethyl, pyridylethyl, piperidinylmethyl, pyrrolidinylmethyl,morpholinylmethyl, quinolylmethyl, etc. Such groups may be substitutedby one or more halogens, hydroxyl groups, lower alkyl groups, or loweralkoxy groups (including combinations of such groups).

As used herein, “aryl” refers to the typical substituted orunsubstituted non-aliphatic hydrocarbyl groups of this class, i.e., apolyunsaturated, typically aromatic, hydrocarbon substituent, which canbe a single ring or multiple rings (up to three rings) which are fusedtogether or linked covalently, such as phenyl, naphthyl, and the like.This class of moieties also includes fused-ring moieties such asindanyl, etc. Substituents for the aromatic moieties are similar tothose for the aliphatic groups. “Aryl”, as used herein, also includesanalogous heterocyclic groups (sometimes termed “heteroaromatic”groups), namely polyunsaturated cyclical moieties containing carbonatoms in the ring and additionally one or more hetero atoms, which aretypically oxygen, nitrogen, sulfur and or phosphorus, such as pyridinyl,pyrazinyl, pyrazolyl, thienyl, furyl, thiazolyl, imidazolyl, pyrrolyl,etc., and fused-ring moieties such as benzoxazolyl, benzthiazolyl, etc.These may be optionally substituted with one or more substituents suchas halogen, hydroxy, amino, optionally substituted lower alkyl,optionally substituted lower alkoxy, NCONR₃R₄ and NCOOR₃, and othersubstituents included above within the definition of group R₆.

Aryl compounds also include fluorescent aromatic moieties such as

Carbocyclic or heterocyclic moieties for W generally have the formula

and are, for example:

In general, compounds of Formula (I) may be synthesized from a startingnitrobenzene or aminobenzene

followed by iodination and closure to form an indole, and alkylation orthe like to add groups R₁ and R₂ and replacement of the oxazolidinegroup by a group Y. An illustration of such a method is shown below.

The following Tables I and II show representative compounds of theinvention, that can be made by the processes described herein TABLE 1(Compounds of Formula I)

Cmpd. No. R₀ R₁ R₂ Y n 1 CH₃ C₂H₄C₆H₄ n-C₄H₉ COOH 0 2 CH₃ CH₂CH₂OHn-C4H₉ COOH 0 3 CH₃ CH₂CH₂OH CHOH(CH₂)₃— COOH 0 CONH₂ 4 CH₃ C₂H₄C₆H₅C6H₅ COOH 0 5 CH₃ C₂H₄C₆H₅ CHOH(CH₂)₃— COOH 0 CONH₂

TABLE 2 Compounds of Formula (II)

Cmpd. No. R₀ R₁ R₂ Y Z 6 CH₃ n-C₅H₁₁ C₂H₄C₆H₅ COOH CH₂ (Na salt) CH₂ 7C₂H₅ n-C₅H₁₁ C₂H₄C₆H₅ COOH CH₂ (Na salt)

A 6×8 procedure that may be used to prepare compounds of the inventionis shown below. The procedure shows preparation of the AcO— and —COOCH₃analogs of these compounds, which are then converted to the compounds ofthe invention having —OH and —COOH groups, respectively.

Formulation and Administration

Compounds of the invention that inhibit interactions between the PDZdomain of a protein and other proteins can be administered to a patientor subject at doses effective to provide the desired inhibition, or attherapeutically effective doses to prevent, treat, or controlconditions, for example to act as neuroprotecting drugs and anti-tumoragents. Compositions containing the substances are administered to apatient or subject in an amount sufficient to elicit an effectivetherapeutic response in the patient. An amount adequate to accomplishthis is defined as an “effective inhibitory amount,” a “therapeuticallyeffective dose” or a “therapeutically effective amount”. The dose oramount will be determined by the efficacy of the particular activesubstance employed and the condition of the subject. The size of thedose also will be determined by the existence, nature, and extent of anyadverse effects that accompany the administration of a particularcompound in a particular subject. Typically, the patient or subject ishuman. However, the patient or subject may be a non-human mammal (e.g.,a primate, a mouse, a pig, a cow, a cat, a goat, a rabbit, a rat, aguinea pig, a hamster, a horse, a sheep, a dog, a cat and the like), andmay be male or female.

Toxicity and therapeutic efficacy of the compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, for example, by determining the LD₅₀ (the dose lethal to 50% ofthe population) and the ED₅₀ (the dose therapeutically effective in 50%of the population). The dose ratio between toxic and therapeutic effectsis the therapeutic index and can be expressed as the ratio, LD₅₀/ED₅₀.Compounds that exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects can be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue to minimize potential damage to normal cells andthereby reduce side effects.

The data obtained from cell culture assays and animal studies can beused to formulate a dosage range for use in humans. The dosage of suchcompounds lies preferably within a range of circulating concentrationsthat include the ED₅₀ with little or no toxicity. The dosage can varywithin this range depending upon the dosage form employed and the routeof administration. For any compound used in the methods of theinvention, the therapeutically effective dose can be estimated initiallyfrom cell culture assays. A dose can be formulated in animal models toachieve a circulating plasma concentration range that includes the IC₅₀(the concentration of the test compound that achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma can be measured, for example, by high performance liquidchromatography (HPLC).

Pharmaceutical compositions for use in the present invention can beformulated by standard techniques using one or more physiologicallyacceptable carriers or excipients. The compounds and theirphysiologically acceptable salts and solvates can be formulated foradministration by any suitable route, including via inhalation,topically, sublingually, intranasally, orally, parenterally (e.g.,intravenously, intraperitoneally, intramuscularly, subcutaneously,intravesically or intrathecally), or mucosally (including intranasally,orally and rectally).

For oral or sublingual administration, pharmaceutical compositions ofcompositions of the invention can take the form of, for example,lozenges, tablets or capsules prepared by conventional means withpharmaceutically acceptable excipients, including binding agents, forexample, pregelatinized cornstarch, polyvinylpyrrolidone, orhydroxypropyl methylcellulose; fillers, for example, lactose,microcrystalline cellulose, or calcium hydrogen phosphate; lubricants,for example, magnesium stearate, talc, or silica; disintegrants, forexample, potato starch or sodium starch glycolate; or wetting agents,for example, sodium lauryl sulfate. Tablets can be coated by methodswell known in the art. Liquid preparations for oral administration cantake the form of, for example, solutions, syrups, or suspensions, orthey can be presented as a dry product for constitution with water orother suitable vehicle before use. Such liquid preparations can beprepared by conventional means with pharmaceutically acceptableadditives, for example, suspending agents, for example, sorbitol syrup,cellulose derivatives, or hydrogenated edible fats; emulsifying agents,for example, lecithin or acacia; non-aqueous vehicles, for example,almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils;and preservatives, for example, methyl or propyl-p-hydroxybenzoates orsorbic acid. The preparations can also contain buffer salts, flavoring,coloring, and/or sweetening agents as appropriate. If desired,preparations for oral administration can be suitably formulated to givecontrolled release of the active compound.

For administration by inhalation, the compounds may be convenientlydelivered in the form of an aerosol spray presentation from pressurizedpacks or a nebulizer, with the use of a suitable propellant, forexample, dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In thecase of a pressurized aerosol, the dosage unit can be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, for example, gelatin for use in an inhaler or insufflator can beformulated containing a powder mix of the compound and a suitable powderbase, for example, lactose or starch.

The compounds can be formulated for parenteral administration byinjection, for example, by bolus injection or continuous infusion.Formulations for injection can be presented in unit dosage form, forexample, in ampoules or in multi-dose containers, with an addedpreservative. The compositions can take such forms as suspensions,solutions, or emulsions in oily or aqueous vehicles, and can containformulatory agents, for example, suspending, stabilizing, and/ordispersing agents. Alternatively, the active ingredient can be in powderform for constitution with a suitable vehicle, for example, sterilepyrogen-free water, before use.

The compositions of the invention may also be formulated in rectalcompositions such as suppositories or retention enemas, e.g., containingconventional suppository bases such as cocoa butter or other glycerides.

The compositions of the invention may also be formulated for transdermaladministration. For transdermal administration, the active compounds areformulated into ointments, salves, gels, or creams as generally known inthe art. Pharmaceutical compositions adapted for transdermaladministration can be provided as discrete patches intended to remain inintimate contact with the epidermis for a prolonged period of time. Ifthe compositions of the invention are to be administered topically, thecompositions can be formulated in the form of, e.g., an ointment, cream,transdermal patch, lotion, gel, spray, aerosol, solution, emulsion, orother form well-known to one of skill in the art. For non-sprayabletopical dosage forms, viscous to semi-solid or solid forms comprising acarrier or one or more excipients compatible with topical applicationand having a dynamic viscosity preferably greater than water aretypically employed. Suitable formulations include, without limitation,solutions, suspensions, emulsions, creams, ointments, powders,liniments, salves, and the like, which are, if desired, sterilized ormixed with auxiliary agents (e.g., preservatives, stabilizers, wettingagents, buffers, or salts) for influencing various properties, such as,for example, osmotic pressure. Other suitable topical dosage formsinclude sprayable aerosol preparations wherein the active ingredient,preferably in combination with a solid or liquid inert carrier, ispackaged in a mixture with a pressurized volatile (e.g., a gaseouspropellant, such as Freon), or in a squeeze bottle. Moisturizers orhumectants can also be added to pharmaceutical compositions and dosageforms if desired. Examples of such additional ingredients are well-knownin the art. Compositions may also be included in a device fortransdermal delivery such as a skin patch or a more complex device.

The compounds also may be formulated as a depot preparation. Suchlong-acting formulations can be administered by implantation (forexample, subcutaneously or intramuscularly) or by intramuscularinjection. Thus, for example, the compounds can be formulated withsuitable polymeric or hydrophobic materials (for example as an emulsionin an acceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

The compositions may also be in the form of controlled release orsustained release compositions as known in the art, for instance, inmatrices of biodegradable or non-biodegradable injectable polymericmicrospheres or microcapsules, in liposomes, in emulsions, and the like.

The compositions can, if desired, be presented in a pack or dispenserdevice that can contain one or more unit dosage forms containing theactive ingredient. The pack can, for example, comprise metal or plasticfoil, for example, a blister pack. The pack or dispenser device can beaccompanied by instructions for administration.

Depending on their chemical and physical nature, compounds of theinvention may be included in the compositions and administered to thepatient per se, or in another form such as a salt, solvate, complex,chelate or other derivative as appropriate or as needed for goodformulation or administration of the substance. Likewise, a prodrug ofthe substance may be included in the compositions, that is, a substancethat releases the active substance either on preparation of thecomposition or on administration of the composition to the patient orsubject.

As mentioned above, this invention also provides therapeutic methods oftreating cancer comprising administering one or more compounds of theinvention, or a composition containing the same, to the patient. Themethod may further comprise administering to the patient a secondtherapeutic agent, such as a chemotherapeutic agent or radiationtherapy. The cancer being treated may be a breast cancer, a colorectalcancer, a lung cancer, a sarcoma, a mesothelioma, a prostate cancer, apancreatic cancer, a cervical cancer, an ovary cancer, a gastric cancer,an esophageal cancer, a head and neck cancer, a hepatocellularcarcinoma, a melanoma, a glioma, a squamous cancer, or a glioblastoma.

In carrying out the invention, a single inhibitory compound, or acombination of compounds according to this invention may be administeredto a patient. The effective compounds may be administered alone or incombination with (or in time proximity to) other therapeutic agentsadministered for similar or other therapeutic purposes, for exampleadministration of a compound according to this invention together withan adjuvant or other anti-inflammatory agent. Similarly, compositionscontaining one or more of the compounds of this invention may alsocontain other pharmaceutical or therapeutic agents.

The present invention also includes arrays for testing substances forinteraction with or inhibition of PDZ domains. Typically such arrayswill be used for testing combinatorial or other libraries. The arrayswill comprise standard equipment such as a plate, which will containcompounds arranged on the surface of the plate, for example in wells orbound to certain locations on the surface. A plate or array may containcompounds of a single type or it may contain different compounds,located in prearranged fashion.

In one aspect therefore the invention provides in vitro, ex vivo, and invivo assays for inhibitors of proteins having PDZ domains or forstudying inhibition of the activity of a given PDZ domain or a proteincontaining one. In particular, the assays can be used to test forcompounds that possess this activity for testing for binding to, orinhibition of the activity of, PDZ domains. Typically in such assays,the compound or compounds to be tested are contacted with the proteinhaving a PDZ domain and suitable tests are carried out to ascertainwhether the normal activity of that domain has been inhibited. Forexample, the results of the assay may be compared to a control assaythat comprises the protein alone, without the test compound(s), usingany known activity of the protein as the comparison standard.

Alternatively screening of a compound for inhibition of PDZ domainactivity of a protein may comprise contacting such a protein or a cellcontaining or expressing it with a compound of the invention anddetecting specific binding of the compound to the domain. The detectingmay be carried out via a method such as capillary electrophoresis,Western blot, mass spectroscopy, ELISA, immunochromatography, orimmunohistochemistry.

Binding of test compounds to PDZ domains of proteins can be performed insolution, in a bilayer membrane, attached to a solid phase, in a lipidmonolayer, or in vesicles. Binding of test compounds can be tested bymeasuring or observing changes in activity or by, e.g., changes inspectroscopic characteristics or in chromatographic or solubilityproperties. Binding of test compounds can also be ascertained incompetitive binding assays, for example, by ascertaining whetherunlabeled test compounds prevent the interaction between the protein anda biotinylated or fluorescent derivative of a reference compound.

The assays that form an aspect of this invention may be designed toscreen large chemical libraries for inhibition of one or more of theproteins using automated assay steps, which are typically run inparallel (e.g., in microtiter formats on microtiter plates in roboticassays). In one preferred embodiment, high throughput screening methodsare used that involve providing a combinatorial chemical or otherlibrary containing a large number of potential inhibitory compounds.Such libraries are then screened in one or more assays, as describedherein, to identify those library members (either particular chemicalspecies or subclasses) that display the desired activity. When screeningfor modulators, a positive assay result need not indicate thatparticular test agent is a good pharmaceutical. Rather, a positive testresult can simply indicate that the test agent can be used to inhibitactivity of a PDZ domain of a protein. The compounds thus identified mayserve as conventional “lead compounds” for PDZ domain inhibitordiscovery or may themselves be used as potential or actual therapeutics.

Thus, another aspect of this invention lies in libraries, such ascombinatorial libraries, of compounds that are produced for testingbased on activity, i.e., inhibition of a PDZ domain as described herein,within the general definitions of compounds herein, such as formulas(I)-(II). A combinatorial chemical library is a collection of suchchemical compounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library is formedby combining a set of chemical building blocks in every possible way fora given compound type.

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.).

Chemical libraries containing a multiplicity of compounds of Formulas(I) or (II), respectively, are an aspect of this invention and may besynthesized by conducting parallel syntheses as described above forindividual compounds.

The compounds used in screening or testing for inhibition of PDZ domainfunctioning are typically supported on a solid inert support, which maybe particulate or non-particulate. Typically the immobilization isachieved by covalently or otherwise bonding the compounds to the solidsupport material. The bond may be made via a moiety that is part of thechemical composition of the support or that is attached to it, forexample to provide an activated surface (for instance, in the case ofglass). Numerous types of solid supports suitable for immobilizingcompounds are known in the art. These include nylon, nitrocellulose,activated agarose, diazotized cellulose, latex particles, plastic,polystyrene, glass and polymer coated surfaces. These solid supports areused in many formats such as membranes, microtiter plates, beads,probes, dipsticks etc. A wide variety of chemical procedures are knownto covalently link various compounds directly or through a linker tothese solid supports.

Typically the use of any solid support requires the presence of anucleophilic group to react with a compound that must contain a“reactive group” capable of reacting with the nucleophilic group.Alternatively, a “reactive group” is present or is introduced into thesolid support to react with a nucleophile present in or attached to theoligonucleotide. Suitable nucleophilic groups or moieties includehydroxyl, sulfhydryl, amino and activated carboxyl groups, while thegroups capable of reacting with these and other nucleophiles (reactivegroups) include dichlorotriazinyl, alkylepoxy, maleimido, bromoacetylgroups and others. Chemical procedures to introduce the nucleophilic orthe reactive groups onto solid support are known in the art, and includeprocedures to activate nylon (U.S. Pat. No. 5,514,785), glass (Rodgerset al., Anal. Biochem., 23-30 (1999)), agarose (Highsmith et al., J.,Biotechniques 12: 418-23 (1992) and polystyrene (Gosh et al., Nuc. AcidRes., 15: 5353-5372 (1987)). Dependent on the presence of either areactive or nucleophilic groups on the solid support and in the testcompound, coupling can either be performed directly or with bifunctionalreagents. Bifunctional and coupling reagents are well known in the artand many are available from commercial sources.

Typically, glass surfaces are activated by the introduction of amino-,sulfhydryl-, carboxyl- or epoxyl-groups to the glass using theappropriate siloxane reagent. Specifically, immobilization ofoligonucleotide arrays on glass supports has been described: by Guo etal., Nuc. Acid Res., 22: 5456-5465 (1994) using 1,4-phenylenediisothiocyanate; by Joos et al., Anal. Biochem., 247: 96-101 (1997)using succinic anhydride and carbodiimide coupling; and by Beatti, etal., Mol. Biotech., 4: 213-225 (1995) using3-glycidoxypropyltrimethoxysilane.

The following are examples of the preparation of compounds according tothis invention.

Synthesis of PDZ domain inhibitors[1-(2-hydroxyethyl)-2-(1-Hydroxypentyl)-5-methyl]indole-6-carboxylicacid (2) and[2-(1-Hydroxypentyl)-5-methyl-1-(2-phenylethyl)]indole-6-carboxylic acid(3) [Formula (I) Table 1, compounds 2 and 1, respectively]

The general scheme for these syntheses was:

a) HOCH₂C(CH₃)₂NH₂ (3 eq), HBTU (1.2 eq), DIPEA (2 eq), DMF, 40° C., 14hr. b) SOCl₂ (5 eq), CH₂Cl₂, rt, 0.5 hr. c) Fe (7.8 eq), NH₄Cl (aq),EtOH, reflux, 3 hr, 92% over 3 steps. d) ICl (1.6 eq), CaCO₃ (30 eq),MeOH, H₂O, rt, 1 hr. 9a: 36%, 9b: 47%. e) H₂, Pd—C, MeOH, Et₃N, rt, 1hr, quant. f) Ethyl pyruvate (5 eq), Pd(OAC)₂ (0.2 eq), DABCO (5 eq),DMF, 105° C., 50 min, 44%. g) LiAlH₄ (10 mol), THF, reflux, 2.5 hr, 75%.h) MnO₂ (1.8 eq), CH₂Cl₂, rt, 12 hr, 76%. i) BrCH₂CH₂Ph (5 eq), K₂CO₃(10 eq), DMF, 40° C., 22 hr, 93%. j) BrCH₂CO₂Et (1.05 eq), NaH (1.1 eq),DMF, 0° C., 10 min, 74%. k) LiAlH₄ (20 eq), THF, reflux, 0.5 hr, 58%. l)MnO₂ (19 eq), CH₂Cl₂, rt, 12 hr, 84%. m) n-BuMgBr (5 eq), 0° C., 0.5 hr,14a: 72%, 14b: 87%. n) i. MeI (large excess), K₂CO₃ (3 eq), acetone, rt,2.5 d; ii. NaOH, H₂O, MeOH, reflux, 7 hr, 2 steps overall 4a: 65%, 4b:89%. o) Mel (large excess), K₂CO₃ (large excess), acetone, rt, 1.5 h,69%. p) H₂, 10% Pd—C, HCl (20 eq), MeOH, rt, 1 hr, 25%.

Scheme for Synthesis of PDZ domain inhibitorSodium[3-hydroxymethyl-2-(1-pentyl)-1-(2-phenylethyl)-5-methyl]indole-6-carboxylate(4) [Formula (II); Table 2, compound 6]

The general scheme for the synthesis of this compound was:

a) MeI (2 eq.), K₂CO₃ (3 eq.), DMF, 40° C., 1 hr. b) H₂, 10% Pd—C, MeOH,rt, 2 hr, 2 steps quant. c) ICl (1.6 eq), CaCO₃ (3 eq), MeOH, H₂O, rt, 1hr, 34%. d) 1-heptyne (5 eq.), PdCl₂ (PPh₃)₂ (0.15 eq.), CuI (0.3 eq.),Et₂NH (large excess), DMF, rt, 2 hr. e) PdCl₂ (PhCN)₂ (0.2 eq.), DMF,80° C., 40 min, 2 steps overall 87%. f) POCl₃ (1.3 eq.), DMF, 5° C.,quant. g) BrCH₂CH₂Ph (10 eq), Cs₂CO₃ (5 eq), DMF, rt, 19 hr, 44%. h)NaBH₄ (excess), MeOH, 5° C. i) NaOH, H₂O, MeOH, 65° C., 12 hr.

2-[(3-Amino-6-methyl)phenyl]-4,4-dimethyloxazoline (8). A mixture of2-methyl-5-nitrobenzoic acid (6, 1.0 g, 4.8 mmol),2-amino-2-methylpropanol (0.64 g, 7.2 mmol), HBTU (2.2 g, 5.8 mmol),N,N-diisopropylethylamine (1.7 mL, 9.7 mmol), and DMF (5 mL) was stirredat 40° C. for 2 hours. Additional 2-amino-2-methylpropanol (0.64 g, 7.2mmol) was added and stirred at 40° C. for 12 hours. The reaction mixturewas diluted with ethyl acetate (50 mL) and washed with water twice (50mL each) followed by brine (50 mL), dried (Na₂SO₄), and evaporated togive amide as pale-brown solid: ¹H NMR (CDCl₃, 400 MHz) δ 8.19 (d, J=2.4Hz, 1H), 8.17 (dd, J=2.4 Hz and 8.4 Hz, 1H), 7.39 (d, J=8.4 Hz, 1H),5.96 (broad s, 1H), 3.98 (broad s, 1H), 3.73 (s, 2H), 2.54 (s, 3H), 1.44(s, 6H): ¹³C NMR (CDCl₃, 100 MHz) δ 168.74, 145.98, 144.12, 137.94,132.22, 124.69, 121.85, 70.42, 57.21, 24.65, 20.12: EIHRMS m/z found:221.0928 (M-CH₂OH, calcd. for C₁₁H₁₃N₂O₃: 221.0926). The crude amide wastreated with thionyl chloride dichloromethane solution (2 M, 12 mL) atroom temperature for 0.5 hours. The reaction mixture was poured into ice(50 g), added 10% aqueous sodium hydroxide solution (30 mL), extractedwith ethyl acetate twice (40 mL each). The organic layer was washed withwater twice (50 mL each) followed by brine (50 mL), dried (Na2SO4), andevaporated to give oxazoline 7 as a pale-brown oil. 7: 1H NMR (CDCl₃,400 MHz) δ 8.60 (d, J=2.4 Hz, 1H), 8.13 (dd, J=2.4 Hz and 8.4 Hz, 1H),7.37 (d, J=8.4 Hz, 1H), 4.08 (s, 2H), 2.66 (s, 3H), 1.38 (s, 6H): ¹³CNMR (CDCl₃, 100 MHz) δ 160.45, 146.73, 146.10, 132.29, 129.06, 125.20,124.89, 78.97, 68.76, 28.64, 22.14: EIHRMS m/z found: 234.1004 (calcdfor C₁₂H₁₄N₂O₃: 234.1004). A mixture of crude 7, ethanol (5 mL),saturated aqueous ammonium chloride solution (0.5 mL), and iron powder(1.3 g) was heated under reflux for 1 hour. Additional iron powder (0.8g) was added to the reaction mixture and continued the heating for 2hours. The reaction mixture was filtered and was diluted with ethylacetate (50 mL) and washed with water twice (50 mL each) followed bybrine (50 mL), dried (Na₂SO₄), and evaporated to give 8 (896 mg, 92%).8: reddish oil, ¹H NMR (CDCl₃, 400 MHz) δ7.11 (d, J=2.4 Hz, 1H), 6.99(d, J=7.6 Hz, 1H), 6.67 (dd, J=2.4 Hz and 8.0 Hz), 4.05 (s, 2H), 3.6(broad s, 2H), 2.42 (s, 3H), 1.37 (s, 6H): ¹³C NMR (CDCl₃, 100 MHz)δ163.30, 144.06, 132.05, 128.22, 128.20, 117.66, 116.45, 78.81, 67.68,28.58, 20.48: EIHRMS m/z found: 204.1270 (calcd for C₁₂H₁₆N₂O:204.1263).

2-[(3-Amino-4-iodo-6-methyl)phenyl]-4,4-dimethyloxazoline (9a) and2-[(3-Amino-2-iodo-6-methyl)phenyl]-4,4-dimethyloxazoline (9b). Iodinemonochloride dichloromethane solution (1 M, 4.1 mL) was added to amixture of 8 (522 mg, 2.56 mmol), methanol (5 mL), water (0.5 mL), andcalcium carbonate (0.78 g, 7.8 mmol) at 0° C. and stirred at roomtemperature for 1 hour. The reaction mixture was stirred with a solutionof sodium sulfite (0.53 g) in water (2 mL) at room temperature for 1hour, filtered and concentrated under reduced pressure. The concentratedsolution was diluted with ethyl acetate (30 mL) and washed with watertwice (30 mL each) followed by brine (30 mL), dried (Na₂SO₄), andevaporated. The residue was purified with flash chromatography (silicagel, 2:1 n-hexane/ethyl acetate) to give 9a (304 mg, 36%) and 9b (394mg, 47%). 9a: pale-brown solid, ¹H NMR (CDCl₃, 400 MHz) δ 7.51 (s, 1H),7.16 (s, 1H), 4.05 (s, 2H), 3.99 (broad s, 2H), 2.39 (s, 3H), 1.36 (s,6H): ¹³C NMR (CDCl₃, 100 MHz) δ162.56, 144.73, 141.19, 129.68, 128.69,115.71, 87.28, 78.88, 67.87, 28.60, 20.17: EIHRMS m/z found: 330.0220(calcd for C₁₂H₁₅IN₂O: 330.0229). 9b: pale-brown solid, ¹H NMR (CDCl₃,400 MHz) δ 6.96 (d, J=8.4 Hz), 6.69 (d, J=8.0 Hz), 4.15 (s, 2H), 4.06(broad s, 2H), 2.27 (s, 3H), 1.45 (s, 6H).

Ethyl [6-(4,4-dimethyloxazolin-2-yl)-5-methyl]indole-2-carboxylate (10).A mixture of 9a (2.48 g, 7.51 mmol), ethyl pyruvate (4.2 mL, 38 mmol),DABCO (4.2 g, 38 mmol), palladium acetate (337 mg, 1.50 mmol) and DMF(22.5 mL) was stirred at 105° C. for 50 minutes. The reaction mixturewas diluted with ethyl acetate (100 mL) and washed with water twice (100mL each) followed by brine (100 mL), dried (Na₂SO₄), and evaporated. Theresidue was purified with flash chromatography (silica gel, 2:1n-hexane/ethyl acetate) to give 10 (985 mg, 44%). 10: pale-brown solid,¹H NMR (CDCl₃, 400 MHz) δ 8.85 (s, 1H), 7.86 (s, 1H), 7.50 (s, 1H), 7.12(s, 1H), 4.41 (quartet, J=6.8 Hz, 2H), 4.10 (s, 2H), 2.62 (s, 3H), 1.41(s, 6H), 1.41 (t, J=4.4 Hz): ¹³C NMR (CDCl₃, 100 MHz) δ163.53, 162.09,135.01, 130.25, 129.47, 129.28, 125.50, 123.87, 114.01, 107.88, 78.84,68.05, 61.34, 28.63, 21.82, 14.56: EIHRMS m/z found: 300.1473 (calcd forC₁₇H₂₀N₂O₅: 300.1474).

[6-(4,4-Dimethyloxazolin-2-yl)-5-methyl]indole-2-carboxaldehyde (5). Toa suspension of lithium aluminum hydride (0.49 g, 12.7 mmol) in THF (1mL), a solution of 10 (766 mg, 2.55 mmol) in THF (6 mL) was added at 0°C. and heated under reflux. After 2 hours, additional suspension oflithium aluminum hydride (0.49 g, 12.7 mmol) in THF (3 mL) was added at0° C. and heated under reflux for 0.5 hours. The reaction mixture wasworked up in a standard manner. The crude product was washed with 5%ethyl acetate in n-hexane and dried to give alcohol (491 mg, 75%):pale-brown solid, ¹H NMR (CDCl₃, 400 MHz) δ 8.28 (s, 1H), 7.68 (s, 1H),7.36 (s, 1H), 6.29 (s, 1H), 4.78 (s, 2H), 4.11 (s, 2H), 2.57 (s, 3H),1.42 (s, 6H): ¹³C NMR (CD₃OD, 100 MHz) δ167.74, 142.97, 135.89, 132.14,128.85, 122.55, 121.80, 113.76, 100.07, 80.06, 68.06, 58.08, 28.50,21.33: EIHRMS m/z found: 258.1362 (calcd for C₁₅H₁₈N₂O₂: 258.1368).Activated manganese oxide (188 mg) was added to a solution of thealcohol (315 mg, 1.22 mmol) in dichloromethane (30 mL), and stirred atroom temperature for 12 hours. The reaction mixture was filtered andevaporated. The residue was washed with n-hexane and dried to give 5(237 mg, 76%). 5: brown solid, ¹H NMR (CDCl₃, 400 MHz) δ9.84 (s, 1H),9.01 (s, 1H), 7.88 (s, 1H), 7.57 (s, 1H), 7.18 (s, 1H), 4.11 (s, 2H),2.62 (s, 3H), 1.42 (s, 6H): ¹³C NMR (CDCl₃, 100 MHz) □ 182.51, 163.27,137.51, 136.33, 130.54, 128.89, 127.44, 124.51, 114.60, 114.07, 78.88,68.17, 28.59, 21.72: EIHRMS m/z found: 256.1202 (calcd for C₁₅H₁₆N₂O₂:256.1212).

[6-(4,4-Dimethyloxazolin-2-yl)-5-methyl-1-(2-phenylethyl)]indole-2-carboxaldehyde(13b). A mixture of 5 (80 mg, 0.31 mmol), 2-bromoethylbenzene (210microL, 1.56 mmol), potassium carbonate (0.43 g, 3.1 mmol) and DMF (2mL) was stirred at room temperature for 22 hours. The reaction mixturewas diluted with ethyl acetate (20 mL) and washed with water twice (10mL each) followed by brine (10 mL), dried (Na₂SO₄), and evaporated. Theresidue was purified with preparative thin-layer chromatography (silicagel, 3:1 n-hexane/ethyl acetate) to give 13 (105 mg, 93%). 13:pale-brown solid, ¹H NMR (CDCl₃, 400 MHz) δ9.82 (s, 1H), 7.70 (s, 1H),7.55 (s, 1H), 7.25-7.15 (m, 6H), 4.77 (t, J=7.2 Hz, 2H), 4.12 (s, 2H),3.04 (t, J=7.2 Hz, 2H), 2.62 (s, 3H), 1.43 (s, 6H): 13C NMR (CDCl₃, 100MHz) □182.60, 163.06, 138.38, 138.27, 136.70, 130.46, 129.15, 128.60,127.91, 127.20, 126.71, 124.61, 116.75, 112.49, 78.81, 68.29, 46.46,37.09, 28.64, 21.74: EIHRMS m/z found: 360.1843 (calcd for C₂₃H₂₄N₂O₂:360.1838).

1-[[6-(4,4-Dimethyloxazolin-2-yl)-5-methyl-1-(2-phenylethyl)]indol-2-yl]pentanol(14b). To a solution of 13 (54 mg, 0.15 mmol) in dry THF (0.5 mL),n-butylmagnesium bromide THF solution (1 M, 0.45 mL) was added at 0° C.under argon atmosphere and stirred for 0.5 hours. The reaction mixturewas quenched with water (2 mL), diluted with ethyl acetate (10 mL),washed with water twice (10 mL each) followed by brine (10 mL), dried(Na₂SO₄), and evaporated. The residue was purified with preparativethin-layer chromatography (silica gel, 2:1 n-hexane/ethyl acetate) togive 14 (54 mg, 87%). 14: pale-brown oil, ¹H NMR (CDCl₃, 400 MHz) δ7.77(s, 1H), 7.39 (s, 1H), 7.25-7.21 (m, 3H), 6.98-6.96 (m, 2H), 6.28 (s,1H), 4.50-4.41 (m, 1H), 4.40-4.30 (m, 1H), 4.23 (t, J=7 Hz, 1H), 4.11(s, 2H), 3.07 (t, J=7.2 Hz, 1H), 2.63 (s, 3H), 1.84-1.6 (m, 2H), 1.43(s, 6H), 1.4-1.2 (m, 4H), 0.88 (t, J=6.8 Hz, 3H): ¹³C NMR (CDCl₃, 100MHz) δ164.38, 145.18, 138.92, 134.73, 129.75, 129.05, 128.96, 128.68,126.73, 122.46, 121.32, 111.56, 98.31, 78.71, 67.82, 66.43, 45.18,36.43, 28.71, 28.48, 22.68, 21.95, 21.52, 14.22: EIHRMS m/z found:418.2628 (calcd for C₂₇H₃₄N₂O₂: 418.2620).

[2-(1-Hydroxypentyl)-5-methyl-1-(2-phenylethyl)]indole-6-carboxylic acid(3). A mixture of 14 (54 mg, 0.13 mmol), acetone (0.5 mL), podomethane(1 mL) and potassium carbonate (54 mg, 0.39 mmol) was stirred at roomtemperature for 2.5 days, filtered and evaporated. The residue wasdissolved with methanol (1 mL), added aqueous sodium hydroxide solution(10%, 0.3 mL) and heated under reflux for 7 hours. The reaction mixturewas acidified into pH 6 with hydrochloric acid (1 M), diluted with ethylacetate (10 mL), washed with brine (10 mL), dried (Na₂SO₄), andevaporated. The residue was purified with preparative thin-layerchromatography (silica gel, 2:1 n-hexane/ethyl acetate) to give 3 (42mg, 89%). 3: pale-brown solid, ¹H NMR (CD₃0D, 400 MHz) δ8.04 (s, 1H),7.35 (s, 1H), 7.21-7.15 (m, 3H), 7.00-6.89 (m, 2H), 6.30 (s, 1H), 4.45(t, J=6.8 Hz, 2H), 4.29 (dd, J=2.8 Hz and 5.6 Hz, 1H), 3.11-2.99 (m,2H), 2.64 (s, 3H), 1.86-1.67 (m, 2H), 1.42-1.18 (m, 4H), 0.92 (t, J=6.8Hz, 3H): ¹³C NMR (CDCl₃, 100 MHz) δ172.98, 147.31, 140.20, 136.08,132.29, 131.46, 129.99, 129.55, 127.59, 124.50, 123.59, 114.04, 99.29,66.79, 46.40, 37.43, 36.76, 29.47, 23.64, 22.58, 14.47: EIHRMS m/zfound: 365.1986 (calcd for C₂₃H₂₇NO₅: 365.1991).

Ethyl[6-(4,4-dimethyloxazolin-2-yl)-1-ethoxycarbonylmethyl-5-methyl]indole-2-carboxylate(11). To a solution of 6 (449 mg, 1.50 mmol) in DMF (5 mL), sodiumhydride (60% suspension in mineral oil, 66 mg, 1.65 mmol) was added at0° C. under argon atmosphere. After the solution was stirred at 0° C.for 0.5 hours, ethyl bromoacetate (174 microL, 1.58 mmol) was added andstirred for 10 minutes. The reaction mixture was diluted with ethylacetate (50 mL), washed with water twice (50 mL each) followed by brine(50 mL), dried (Na₂SO₄), and evaporated. The residue was purified withflash chromatography (silica gel, 3:1 n-hexane/ethyl acetate) to give 11(428 mg, 74%). 11: pale-yellow solid, ¹H NMR (CDCl₃, 400 MHz) δ7.76 (s,1H), 7.50 (s, 1H), 7.26 (s, 1H), 5.27 (s, 2H), 4.32 (quartet, J=7.2 Hz,2H), 4.20 (quartet, J=7.2 Hz, 2H), 4.10 (s, 2H), 2.61 (s, 3H), 1.41 (s,6H), 1.38 (t, J=7.2 Hz, 3H), 1.26 (t, J=7.2 Hz, 3H): ¹³C NMR (CDCl₃, 100MHz) δ168.97, 163.53, 162.05, 137.57, 130.64, 129.65, 127.90, 125.59,124.23, 111.68, 110.32, 78.83, 68.00, 61.60, 60.97, 46.49, 28.62, 21.83,14.43, 14.26: EIHRMS m/z found: 386.1849 (calcd for C₂₁H₂₆N₂O₅:386.1842).

[[6-(4,4-Dimethyloxazolin-2-yl)-1-(2-hydroxyethyl)-5-methyl]indol-2-yl]methanol(12) was obtained (40 mg, 58%) from 11 (88 mg, 0.23 mmol) by a similarmanner as described in a procedure for synthesis of 5 except theoxidation using manganese dioxide was omitted. 12: pale-yellow solid, ¹HNMR (CD₃OD, 400 MHz) δ7.71 (s, 1H), 7.36 (s, 1H), 6.37 (s, 1H), 4.76 (s,2H), 4.34 (t, J=5.6 Hz, 2H), 4.18 (s, 2H), 3.86 (t, J=5.6 Hz, 2H), 2.52(s, 3H), 1.39 (s, 6H): ¹³C NMR (CD₃OD, 100 MHz) δ167.63, 143.64, 136.73,131.42, 129.31, 123.13, 122.16, 112.35, 101.87, 80.14, 68.11, 62.80,61.96, 57.22, 28.54, 21.30: EIHRMS m/z found: 302.1630 (calcd forC₁₇H₂₂N₂O₃: 302.1630).

[6-(4,4-Dimethyloxazolin-2-yl)-1-(2-hydroxyethyl)-5-methyl]indole-2-carboxaldehyde(13a) was obtained (33 mg, 84%) from 12 (40 mg, 0.13 mmol) by a similarmanner as described in a procedure for synthesis of 5 except thereduction using lithium aluminum hydride was omitted. 13a: pale-yellowoil, ¹H NMR (CDCl₃, 400 MHz) δ9.80 (s, 1H), 7.88 (s, 1H), 7.52 (s, 1H),7.16 (s, 1H), 4.62 (t, J=5.2 Hz, 2H), 4.11 (s, 2H), 3.90 (t, J=5.2 Hz,2H), 2.58 (s, 3H), 1.40 (s, 6H): ³C NMR (CDCl₃, 100 MHz) δ183.23,163.78, 139.14, 136.93, 130.40, 127.98, 127.21, 124.63, 117.33, 113.05,79.11, 67.96, 62.39, 47.15, 28.57, 21.60: EIHRMS m/z found: 300.1474(calcd for C₁₇H₂₀N₂O₃: 300.1474).

1-[[6-(4,4-Dimethyloxazolin-2-yl)-1-(2-hydroxyethyl)-5-methyl]indol-2-yl]pentanol(14a) was obtained (28 mg, 72%) from 13a (33 mg, 0.11 mmol) by a similarmanner as described in a procedure for synthesis of 14b. 14a:pale-yellow oil, ¹H NMR (CDCl₃, 400 MHz) δ7.67 (s, 1H), 7.39 (s, 1H),6.37 (s, 1H), 4.79 (t, J=6.8 Hz, 1H), 4.42-4.37 (m, 1H), 4.34-4.27 (m,1H), 4.09 (s, 2H), 3.98-3.86 (m, 2H), 2.59 (s, 3H), 2.04-1.95 (m, 2H),1.6-1.48 (m, 2H), 1.45-1.36 (m, 2H), 1.39 (s, 6H), 0.93 (t, J=7.2 Hz,3H): ¹³C NMR (CDCl₃, 100 MHz) δ165.41, 145.54, 134.84, 130.02, 129.02,122.55, 121.16, 111.38, 98.64, 79.11, 67.34, 65.38, 60.58, 45.41, 35.14,28.53, 28.49, 22.90, 21.55, 14.23: EIHRMS m/z found: 358.2259 (calcd forC₂₁H₃₀N₂O₃: 358.2256).

[1-(2-hydroxyethyl)-2-(1-Hydroxypentyl)-5-methyl]indole-6-carboxylicacid (2) was obtained (3.3 mg, 65%) from 14a (6.0 mg, 0.017 mmol) by asimilar manner as described in a procedure for synthesis of 3. 2:pale-brown amorphous, ¹H NMR (CD₃OD, 400 MHz) δ8.05 (s, 1H), 7.37 (s,1H), 6.39 (s, 1H), 4.91 (hidden by solvent peak, supposed as 1H),4.49-4.42 (m, 1H), 4.40-4.32 (m, 1H), 3.86 (t, J=5.6 Hz, 2H), 2.62 (s,3H), 2.02-1.95 (m, 2H), 1.6-1.3 (m, 4H), 0.95 (t, J=6.8 Hz, 3H): ¹³C NMR(CDCl₃, 100 MHz) δ172.93, 147.64, 136.56, 132.30, 131.55, 126.52,123.48, 113.99, 99.50, 66.81, 61.93, 46.71, 36.82, 29.52, 23.69, 22.44,14.42 : EIHRMS m/z found: 305.1627 (calcd for C₁₇H₂₃NO₄: 305.1627).

Methyl(3-amino-4-iodo-6-methyl)benzoate (17). A mixture of 6 (2.0 g, 9.6mmol) iodomethane (1.2 mL), potassium carbonate (4.0 g) and DMF (10 mL)was stirred at 40° C. for 1 hour. The reaction mixture was diluted withethyl acetate (100 mL) and washed with water twice (100 mL each)followed by brine (100 mL), dried (Na₂SO₄), and evaporated to give ester15. A mixture of 15, methanol (40 mL) and 10% palladium on charcoal (0.1g) was stirred under hydrogen atmosphere at room temperature for 2hours. The reaction mixture was filtered and evaporated to give 16 (1.88g, 2 steps quant.). Iodine monochloride dichloromethane solution (1 M,13.1 mL) was added to a mixture of 16 (1.58 g), methanol (16 mL), water(1.6 mL), and calcium carbonate (2.45 g) at 0° C. and stirred at roomtemperature for overnight. The reaction mixture was quenched with asolution of sodium sulfite (1 g) in water (4 mL) at room temperature for1 hour, filtered and concentrated under reduced pressure. Theconcentrated solution was diluted with ethyl acetate (700 mL) and washedwith water twice (50 mL each) followed by brine (30 mL), dried (Na₂SO₄),and evaporated. The residue was purified with flash chromatography(silica gel, 20:1 to 9:1 n-hexane/ethyl acetate) to give 17 (0.81 g,34%). 17: pale-brown solid. Mp 77° C., ¹H NMR (CDCl₃, 400 MHz) δ 7.53(s, 1H), 7.29 (s, 1H), 4.04 (s, 2H), 3.86 (s, 3H), 2.42 (s, 3H): ¹³C NMR(CDCl₃, 100 MHz).

Methyl(2-pentyl-5-methyl)indole-6-carboxylate (19). A mixture of 17 (4.4mg, 0.015 mmol), 1-heptyne (10 μL), diethylamine (15 μL),dichlorobis(triphenylphosphino)palladium (1.6 mg), copper iodide (0.9mg) and degassed DMF (60 μL) was stirred at room temperature under argonatmosphere for 2 hours. The reaction mixture was diluted withdichloromethane (2 mL) and purified with preparative thin-layerchromatography (silica gel, 5:1 n-hexane/ethyl acetate) to givemethyl[3-amino-4-(hept-1-ynyl)-6-methyl]benzoate 18. 18: pale-yellowamorphous, ¹H NMR (CDCl₃, 400 MHz) δ 7.27 (s, 1H), 7.11 (s, 1H), 4.10(s, 2H), 3.85 (s, 3H), 2.46 (t, J=7.2 Hz, 2H), 2.42 (s, 3H), 1.63(quintet, J=7.2 Hz, 2H), 1.44 (quintet, J=7.2 Hz, 2H), 1.37 (quintet,J=7.2 Hz, 2H), 0.92 (t, J=7.2 Hz, 3H). The obtained 18 was dissolvedwith degassed DMF (80 μL), dichlorobis(benzonitrile)palladium (1.1 mg)was added and stirred at 80° C. under argon atmosphere for 0.5 hours.The reaction mixture was diluted with dichloromethane (2 mL) andpurified with preparative thin-layer chromatography (silica gel, 5:1n-hexane/ethyl acetate) to give 19 (3.4 mg, 87% from 17). 19: brownamorphous, ¹H NMR (CDCl₃, 400 MHz) δ 7.99 (broad s, 1H), 7.98 (s, 1H),7.34 (s, 1H), 6.18 (s, 1H), 3.89 (s, 3H), 2.75 (t, J=7.2 Hz, 2H), 2.66(s, 3H), 1.72 (t, J=7.2 Hz, 2H), 1.39-1.34 (m, 4H), 0.90 (t, J=7.2 Hz,3H): ¹³C NMR (CDCl₃, 100 MHz).

Methyl[3-formyl-2-pentyl-1-(2-phenylethyl)-5-methyl]indole-6-carboxylate(21). Phosphorous oxychloride (14 μL) was dissolved with dry DMF (0.5mL) at ice-bath temperature and was kept at the temperature for 0.5hours under argon atmosphere. The solution was added to a solution of 19(30 mg, 0.12 mmol) in dry DMF (0.5 mL) at ice-bath temperature and waskept at the temperature for 50 minutes under argon atmosphere. Thereaction mixture was diluted with ethyl acetate (20 mL), washed with 10%aqueous sodium bicarbonate solution twice (10 mL each) followed by brine(10 mL), dried (Na₂SO₄), and evaporated. The solidified residue wastriturated with 5% ethyl acetate/n-hexane and dried to give 20 as apale-brown solid (33 mg, 99%). Mp 179° C. ¹H NMR (CD₃OD, 400 MHz) δ10.04 (s, 1H), 8.01 (s, 1H), 7.97 (s, 1H), 3.89 (s, 3H), 3.11 (t, J=8.0Hz, 3H), 2.65 (s, 3H), 1.86-1.78 (m, 2H), 1.45-1.35 (m, 2H), 0.92 (t,J=7.2 Hz, 3H): ¹³C NMR (CD₃OD, 100 MHz)

A mixture of 20 (7.1 mg), dry DMF (0.25 mL), 2-bromoethylbenzene (17 μL)and cesium carbonate (16 mg) was stirred at room temperature for 17hours. Additional 2-bromoethylbenzene (17 μL) and cesium carbonate (64mg) was added and stirred at room temperature for 2.5 hours. Thereaction mixture was diluted with ethyl acetate (20 mL), washed withwater followed by brine (10 mL each), dried (Na₂SO₄), and evaporated.The residue was purified with preparative thin-layer chromatography(silica gel, 3:1 n-hexane/ethyl acetate) to give 21: pale-yellowamorphous, ¹H NMR (CDCl₃, 400 MHz) δ 10.06 (s, 1H), 8.16 (s, 1H), 7.99(s, 1H), 7.26-7.22 (m, 3H), 6.98-6.94 (m, 2H), 4.36 (t, J=6.8 Hz, 2H),3.95 (s, 3H), 3.11 (t, J=7.2 Hz, 2H), 2.72 (s, 3H), 2.66 (t, J=8.0 Hz,2H), 1.59-1.50 (m, 2H), 1.34-1.25 (m, 4H), 0.88 (t, J=6.4 Hz, 3H): ¹³CNMR (CDCl₃, 100 MHz) δ168.60, 154.87, 137.64, 135.00, 134.18, 129.09,128.94, 127.39, 124.61, 123.58, 119.13, 113.53, 112.92, 52.05, 45.49,36.18, 31.77, 30.27, 29.90, 24.33, 22.43, 14.07. ESI-HRMS calcd.;391.2147 found; 391.2144.

Sodium[3-hydroxymethyl-2-(1-pentyl)-1-(2-phenylethyl)-5-methyl]indole-6-carboxylate(4). A solution of 21 (4.5 mg, 0.011 mmol) of methanol (0.5 mL) wastreated with sodium borohydride (1 mg). The reaction mixture wasquenched with water (2 mL), diluted with ethyl acetate (10 mL), washedwith water twice (10 mL each) followed by brine (10 mL), dried (Na₂SO₄),and evaporated to givemethyl[3-hydroxymethyl-2-(1-pentyl)-1-(2-phenylethyl)-5-methyl]indole-6-carboxylate22 as a pale-brown amorphous. 22: ¹H NMR (CDCl₃, 400 MHz) δ7.99 (s, 1H),7.49 (s, 1H), 7.30-7.22 (m, 3H), 7.06 (d, J=6.8 Hz, 2H), 4.77 (s, 2H),4.31 (t, J=7.6 Hz, 2H), 3.93 (s, 3H), 3.04 (t, J=7.6 Hz, 2H), 2.70 (s,3H), 2.54 (t, J=7.6 Hz, 2H), 1.56-1.47 (m, 2H), 1.36-1.22 (m, 4H), 0.88(t, J=6.8 Hz, 3H): ¹³C NMR (CDCl₃, 100 MHz) δ169.11, 143.48, 138.43,133.88, 131.52, 130.85, 128.96, 128.89, 127.05, 122.47, 120.49, 112.77,111.05, 55.95, 51.83, 45.37, 36.78, 31.90, 30.31, 24.52, 22.62, 22.58,14.16. HRMS: calcd. 393.2304 found; 393.2314. The obtained 22 wastreated with sodium hydroxide (aqueous 5% solution, 33 μL) in methanol(0.1 mL) at 60° C. for 22 h, evaporated and pumped-up to afford 4 (11mg). 4: white powder, ¹H NMR (CD₃OD, 400 MHz) δ7.64 (s, 1H), 7.40 (s,1H), 7.26-7.19 (m, 3H), 7.09 (d, J=7.2 Hz, 2H), 4.69 (s, 2H), 4.30 (t,J=7.2 Hz, 2H), 3.04 (t, J=7.2 Hz, 2H), 2.60 (s, 3H), 2.52 (t, J=7.6 Hz,2H), 1.54-1.46 (m, 2H), 1.36-1.28 (m, 4H), 0.90 (t, J=6.4 Hz, 3H): ¹³CNMR (CD₃OD, 100 MHz) δ168.14, 141.81, 140.24, 135.15, 135.05, 129.96,129.70, 129.60, 127.88, 127.60, 120.30, 111.25, 110.13, 55.76, 46.27,37.34, 32.91, 31.19, 24.24, 23.45, 21.38, 14.38. ESI-HRMS: calcd.;362.2115 (M−OH)⁺. found; 362.2120 (M−OH)⁺.

Protein Expression

A plasmid consisting of a GST fusion construct of the second PDZ domainof MAGI-3 was obtained from Genentech. The plasmid was transformed intoBL21 DE3 cells, grown at 37° C. to an O.D.₆₀₀ of 0.8, and induced with 1mM of IPTG. After three hours the cells were harvested, lysed, and theprotein was purified through a slurry of glutathione sepharose beads(Pharmacia). The protein was dialyzed into the assay buffer (35 mM HEPESat pH 7.4, 0.01% triton X-100, 10% glycerol) and concentrated with10,000 MW cutoff Centricon filters (Waters). Protein purity was verifiedby SDS-PAGE with Coomassie and silver staining. Quantification of theprotein was done with the BCA protocol by Pierce. The protein was storedin the assay buffer at −80° C. GST alone was expressed, purified andstored in the same manner.

Binding Assay

A fluorescence polarization competition assay was used to detect bindingof the compounds to MAGI-3 PDZ2. A fluorescein labeled carboxy terminalsequence of PTEN, OregonGreen™-PFDEDQHTQITKV-COOH, was used as a probe.For a positive control, we chose PFDEDQHTQITWV-COOH, the highestaffinity peptide sequence for MAGI-3 PDZ2 known. To synthesize thelabeled peptide we used standard Fmoc conditions on Wang resin to builda 13 residue peptide. A typical coupling cycle includes deprotecting theterminal amino acid with 20% piperidine in dry DMF, washing the resin2-3 times with DMF then methylene chloride and both again, anddetermining the existence of free amine by ninhydrin kaiser test. Aslurry containing coupling reagent 2.4 equivalents of HBTU, the next Nterminal amino acid to be added to the growing peptide 2.5 equivalentsof Fmoc protected amino acid, 5 equivalents of DIEA in dry DMF. Theamino acid was coupled over 2-3 hours and the kaiser test was used todetermine completeness. Coupling steps were repeated if a positivekaiser test resulted. This method was used for each residue of thepeptide. The finished peptide was cleaved from the resin with 95% TFAwith a cocktail of scavengers including thioanisole and phenol. Thepeptide was precipitated with ether and lyophilyzed. Peptides werepurified using HPLC and identified with MALDI mass spectrometry.

To detect binding of compounds to the PDZ2 domain of the MAGI-3 protein,a competition polarization assay was employed. The buffer included 35 MMHEPES at pH 7.4, triton X-100 0.01%, and 10% glycerol. The protein wasadded to a final concentration of 300 nM and the probe, 10 nM. Thecompetitor was then added to final concentration range of 10 ρM to 300μM. Triplicates of the samples in a total volume of 20 μL weretransferred to 384 well Corning opaque plate for analysis. Fluorescencepolarization was measured at equilibrium by LJL Biosystems plate reader.Competition data was fit to a one-site competition expression and theIC₅₀ values of the nonpeptide compounds were compared.

FIG. 2 shows the fluorescence polarization competition of inhibitor 3(above) (Table I, compound 1) for the binding site ofOG-PFDEDQHTQITTV-COOH (10 nM) on GST-MAGI-3 PDZ2 (300 nM).

FIG. 3 shows the results of this test: the effect of PDZ inhibitors 2and 4 (above) (Table I, compound 1; Table II compound 6, respectively)on PKB activity.

FIG. 4 shows the results of an evaluation of the irreversible characterof the binding of inhibitor 4 (Table 2, compound 6) to the MAGI3 PDZ2domain

Antibodies

Anti-phospho-Ser473 for immunoblotting were produced by injectingrabbits with the peptides CRPHFPQFS(P)YSASGT, and antibodies recognizingunphosphorylated peptide Anti-phospho-Ser473 for immunoblotting wereproduced by injecting rabbits with the peptides CRPHFPQFS(P)YSASGT, andantibodies recognizing unphosphorylated peptide were removed by bindingto nonphosphopeptide columns. The unbound material was then affinitypurified over a phosphopeptide column. The antibodies used for PKBimmunoprecipitation (IP) kinase assays were generated by injectingrabbits with recombinant full-length PKB. Polyclonal anti-SHIP-2antibody was generated by immunizing rabbits with glutathioneS-transferase fused to the C-terminal region of SHIP-2. Anti-PKB kinase(i.e., PDK-1) was purchased from Transduction Laboratories. Horseradishperoxidase-conjugated secondary antibodies were obtained fromAmersham-Pharmacia.

Immunoprecipitation and in vitro PKB Assay

Subconfluent monolayers of HCT116 cells were lysed by scraping the cellsinto lysis buffer (20 mM Tris-HCl, pH 7.5, containing 1% NP-40, 1 mMEGTA, 1 mM EDTA, 1 mM sodium orthovanadate, and protease inhibitorcocktail [Boehringer Mannheim]) at 4° C. After centrifugation (10,000×gfor 10 min at 4° C.) to remove insoluble components, endogenous PKB wasimmunoprecipitated (IPed) using the anti-PKB antibody and proteinA-Sepharose at 4° C. for 1 h. After washing the IP, kinase activity wasassayed using the synthetic peptide GRPRTSSFAEG (Crosstide) as asubstrate in a reaction mixture containing 20 mM Tris-HCl (pH 7.5), 75mM NaCl, 10 mM MgCl₂, 1 mM dithiothreitol (DTT), 20 μM ATP, 50 μMCrosstide, and 5 μCi of [−³²P]ATP in a volume of 20 μl per assay. Thereaction was allowed to proceed for 15 min at 30° C. and then wasstopped by spotting 18 μl onto Whatman P81 filter papers and immersingthem in 1% (vol/vol) orthophosphoric acid. The papers were washed fourtimes, rinsed once in acetone, and air dried, and the radioactivity wasdetermined by scintillation counting. Alternatively, the phosphorylationreactions were stopped by the addition of Tricine sample buffer, thephosphopeptide was separated on a 16% Tricine gel, and the amount of ³²Pradioactivity was assessed using a STORM PhosphorImager (MolecularDynamics).

Apoptotic Effects of Compounds in Cancer Cells Overexpressing Dvl

Compounds were tested for apoptotic effects to represent the inhibitoryeffects of compounds on interaction between the PDZ domain of theDishevelled (Dlv) protein and the Fz protein (Wnt receptor). Cell linesH513 and H1703 were utilized, with test compounds (compound numbers fromTables 1 and 2) being applied at 0 (control), 10 μM and 100 μM. Resultswere as follows: Compound no. Conc., μM Cell line days apoptosis, % 1 10H1703 4 6.2 1 100 H1703 4 38.7 6 10 H1703 1 8.2 6 100 H1703 1 34.0 6 10H513  1 14.3 6 100 H513  1 52.5

In other tests, Compound 6 (Table II) was found to bind humanDishevelled (Dvl) protein; and at 100 μM, and after 4 days, was alsofound to suppress cancer cell growth and induce apoptosis in

-   -   up to 85.2% of the population in human lung cancer (H1073)        cells;    -   over 50% of the population in human mesothelioma (H513) cells;    -   35% of the population in human melanoma (LOX) cells;    -   about 20% of the population in human lung cancer (H460) cells;        and to inhibit Wnt/beta-Catenin signaling in human lung cancer        cell lines A549 and H1703.

Other proteins that are targets of the compounds of this invention,particularly for anti-tumor activity, include NHREF-1/EPB50, ZO-1, NNOS,ERBIN and MUPP1.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

All publications, patents, and patent applications cited herein arehereby incorporated by reference in their entirety for all purposes.

1. A compound having the formula

in which: R₀ is selected from the group consisting of C₁-C₃ alkyl,cyclopropyl, halo, OR₅ and S(O)_(m)R₅ in which m is 0, 1 or 2; R₁ and R₂are independently selected from the group consisting of C₂-C₈ alkenyl,phenylcyclopropyl, phenylpropenyl, R₆—X₂—C(R₈)(R₈)—R₇—; andR₆—X₂—N(R₈)—R₇—; R₃ and R₄ are independently hydrogen, methyl or ethyl;R₅ is methyl or ethyl; R₆ is selected from the group consisting ofhydrogen, C₁-C₁₀ alkyl, aryl, W, Y, NH₂, NHCONR₃R₄, NHCOOR₃ and NHSO₂R₉;R₇ is selected from the group consisting of a direct bond, an alkylgroup having from 1 to 10 carbon atoms, aryl,—(NH)_(p)(CH₂CH₂O)_(q)(NH)_(p)— in which p is 0 or 1 and q is an integerfrom 1 to 4, and W; R₈ is selected from the group consisting of H, Y,OH, —NHCONR₃R₄; —NHCOOR₃; —NHSO₂R₉, —(CH₂)_(r)CO₂R₃, and(CH₂)_(r)CO₂NR₃R₄ in which r is an integer from 1 to 3; R₉ is aryl orC₁-C₆ alkyl; X₁ is —CH—, —C-hal, —C(CH₃) or —C(C₂H₅), in which halstands for a halogen atom (preferably chloro, fluoro or bromo); X₂ isselected from the group consisting of a direct bond, —NH—, —N(CH₃)—,—NCONR₃R₄—, —NCOOR₃—, and NSO₂R₉; W is a saturated carbocyclic orheterocyclic group; Y is selected from the group consisting of COOH,COOR₃, CONR₃R₄, CONHSO₂R₅, hydroxymethyl, —CH₂COOH, CH₂CONR₃R₄; and5-tetrazolyl; and Z is —CH₂—, —CH(CH₃)—, C(CH₃)₂— or —CO—; and hydratesand salts thereof, and labeled derivatives thereof.
 2. A compound ofFormula (I) according to claim
 1. 3. A compound of Formula (II)according to claim
 1. 4. A compound according to claim 1 in which Y isCOOH or COOR₃.
 5. A compound according to claims 1 in which R₀ is aC₁-C₃ alkyl group.
 6. A compound according to claim 5 in which R₀ ismethyl.
 7. A compound according to claim 2 in which R₁ is optionallysubstituted phenethyl.
 8. A compound according to claim 2 in which R₁ is2-hydroxyethyl.
 9. A compound according to claim 2 in which R₂ isn-butyl, phenyl or n-butyrylamido.
 10. A compound according to claim 2in which R₂ is R₆—X₂—C(R₈)(R₈)—R₇— or R₆—X₂—N(R₈)—R₇—, and the groupR₆—X₂—C(R₈)(R₈)—R₇— or R₆—X₂—N(R₈)—R₇— is selected from C₃-C₈ alkyl;C₃-C₆ cycloalkyl; C₃-C₈ alkenyl; —(CH₂)_(m)C₆H₅ where m is 0 or aninteger from 1-3; —CH₂OC₆H₅, CH₂COC₆H₅, phenyl(C₂-C₄ alkenyl), oranalogous moieties having substituted phenyl groups; optionallysubstituted phenylcyclopropyl; —(CH₂)_(s)OH, —(CH₂)_(s)CONH₂ and—(CH₂)_(s)COOH where s is an integer from 1 to 3; phenyl; thienyl; andoptionally substituted C₃-C₆ cycloalkyl-(C₁-C₃ alkyl).
 11. A compoundaccording to claim 2 in which R₀ is methyl, R₁ is phenethyl, R₂ isn-butyl, X₁ is —CH, Y is COOH and n is
 0. 12. A compound according toclaim 2 in which R₀ is methyl, R₁ is 2-hydroxyethyl, R₂ is n-butyl, X₁is —CH, Y is COOH and n is
 0. 13. A compound according to claim 3 inwhich R₂ is phenethyl or 2-hydroxyethyl.
 14. A compound according toclaim 3 in which R₁ is C₃-C₈ alkyl.
 15. A compound according to claim 3in which R₀ is methyl, R₁ is n-pentyl, R₂ is phenethyl, X₁ is —CH and Yis COOH.
 16. A probe comprising a compound according to claim 1 and adetectable label.
 17. A method for inhibiting the functioning of a PDZdomain of a protein comprising contacting the protein with an inhibitoryeffective amount of a compound according to claim
 1. 18. A method forinhibiting the functioning of a PDZ domain of a protein comprisingcontacting the protein with an inhibitory effective amount of a compoundaccording to claim
 2. 19. A method for inhibiting the functioning of aPDZ domain of a protein comprising contacting the protein with aninhibitory effective amount of a compound according to claim
 3. 20. Amethod for inhibiting the functioning of a PDZ domain of a proteincomprising contacting the protein with an inhibitory effective amount ofa compound according to claim
 7. 21. A method for inhibiting thefunctioning of a PDZ domain of a protein comprising contacting theprotein with an inhibitory effective amount of a compound according toclaim
 8. 22. A method for inhibiting the functioning of a PDZ domain ofa protein comprising contacting the protein with an inhibitory effectiveamount of a compound according to claim
 9. 23. A method for inhibitingthe functioning of a PDZ domain of a protein comprising contacting theprotein with an inhibitory effective amount of a compound according toclaim
 10. 24. A method for inhibiting the functioning of a PDZ domain ofa protein comprising contacting the protein with an inhibitory effectiveamount of a compound according to claim
 11. 25. A method for inhibitingthe functioning of a PDZ domain of a protein comprising contacting theprotein with an inhibitory effective amount of a compound according toclaim
 15. 26. A method according to claim 17 in which the protein is aMAGI protein.
 27. A combinatorial library of two or more compoundshaving the formula

in which: R₀ is selected from the group consisting of C₁-C₃ alkyl,cyclopropyl, halo, OR₅ and S(O)_(m)R₅ in which m is 0, 1 or 2; R₁ and R₂are independently selected from the group consisting of C₂-C₈ alkenyl,phenylcyclopropyl, phenylpropenyl,), R₆—X₂—C(R₈)(R₉)—R₇—; andR₆—X₂—N(R₈)—R₇—; R₃ and R₄ are independently hydrogen, methyl or ethyl;R₅ is methyl or ethyl; R₆ is selected from the group consisting ofhydrogen, C₁-C₁₀ alkyl, aryl, W, Y, NH₂, NHCONR₃R₄, NHCOOR₃ and NHSO₂R₉;R₇ is selected from the group consisting of a direct bond, an alkylgroup having from 1 to 10 carbon atoms, aryl,—(NH)_(p)(CH₂CH₂O)_(q)(NH)_(p)— in which p is 0 or 1 and q is an integerfrom 1 to 4, and W; R₈ is selected from the group consisting of H, Y,OH, —NHCONR₃R₄; —NHCOOR₃; —NHSO₂R₉, —(CH₂)_(r)CO₂R₃, and(CH₂)_(r)CO₂NR₃R₄ in which r is an integer from 1 to 3; R₉ is aryl orC₁-C₆ alkyl; X₁ is —CH—, —C-hal, —C(CH₃) or —C(C₂H₅), in which halstands for a halogen atom (preferably chloro, fluoro or bromo); X₂ isselected from the group consisting of a direct bond, —NH—, —N(CH₃)—,—NCONR₃R₄—, —NCOOR₃—, and NSO₂R₉; W is a saturated carbocyclic orheterocyclic group; Y is selected from the group consisting of COOH,COOR₃, CONR₃R₄, CONHSO₂R₅, hydroxymethyl, —CH₂COOH, CH₂CONR₃R₄; and5-tetrazolyl; and Z is —CH₂—, —CH(CH₃)—, C(CH₃)₂— or —CO—; and hydratesand salts thereof, and labeled derivatives thereof.
 28. A combinatoriallibrary according to claim 27 in which the compounds are of Formula (I).29. A combinatorial library according to claim 27 in which the compoundsare of Formula (II).
 30. A method for screening one or more proteins forPDZ domain activity comprising contacting the one or more proteins witha compound according to claim
 1. 31. An array for screening for PDZdomain activity or inhibition of the same, or for studyingprotein-protein interactions comprising two or more compounds accordingto claim
 1. 32. A method for treating a cancer in cancerous cells or ina patient comprising contacting the cancerous cells with, oradministering to the patient, a therapeutically effective amount of acompound according to claim
 1. 33. A method for treating a cancer in apatient comprising administering to the patient a therapeuticallyeffective amount of a compound according to claim
 2. 34. A method fortreating a cancer in a patient comprising administering to the patient atherapeutically effective amount of a compound according to claim
 3. 35.A method for treating a cancer in a patient comprising administering tothe patient a therapeutically effective amount of a compound accordingto claim
 11. 36. A method for treating a cancer in a patient comprisingadministering to the patient a therapeutically effective amount of acompound according to claim 15.